Discovery and Characterization of Dual Inhibitors of MDM2 and NFAT1 for Pancreatic Cancer Therapy

Pancreatic cancer is a highly aggressive and commonly fatal malignancy, which is characterized by early metastasis and a poor response to chemotherapy (1, 2). Although it demonstrates only modest clinical benefit, gemcitabine remains one of the mainstays of treatment for advanced pancreatic cancer (1). Various multidrug regimens that combine gemcitabine with other chemotherapeutic or molecular targeted agents have been evaluated in the preclinical and clinical settings. However, only three combination regimens have been approved by the FDA, and most of them failed to demonstrate statistically significant benefits on the survival of patients with pancreatic cancer in phase III clinical trials (3–5). Stromal depletion and immunotherapy have also been expected to offer substantial promise for treating advanced pancreatic cancer, but their therapeutic impact remains unclear (6). Thus, there is an unmet clinical need for novel, effective, and safe drugs for pancreatic cancer therapy.

The murine double minute 2 (MDM2) protein is overexpressed in a number of human malignancies, including pancreatic cancer (7, 8). MDM2 exerts its oncogenic activity via both p53-dependent and p53-independent pathways, promoting cancer cell growth and invasion, suppressing apoptosis, and inducing resistance to chemotherapy (7–12). MDM2 overexpression is associated with invasive and high-grade/late-stage tumors, metastasis, and recurrence, and is a negative prognostic factor for cancer patients with pancreatic cancer (7, 8). Recent advances in MDM2 biology have demonstrated that MDM2 is a valid target for cancer therapy (7, 8, 13, 14), and numerous studies using different types of MDM2 inhibitors have shown that inhibition of MDM2 can lead to decreased tumor growth and progression, and an improved response to conventional cancer therapies. Many MDM2 inhibitors have been developed to inhibit the MDM2–p53 interaction, MDM2 expression or its E3 ligase activity, and a few candidates have entered clinical trials. However, none of these inhibitors have been approved for clinical use (12, 14). Of note, almost all of the small molecule inhibitors (SMI) of MDM2 that have been (or are being) tested in clinical trials target the binding of MDM2 and p53, and exert their anticancer effects primarily by activating the p53 pathway in cells with wild-type p53. Considering that these MDM2 SMIs require wild-type p53 expression in cancer cells, they would be expected to have little or no activity against cancers with p53 deficiency or mutations. Unfortunately, genetic alterations of p53 are common in pancreatic cancer, and such cancers are more aggressive and more likely to metastasize (15, 16). Therefore, there is an urgent need to identify a novel, p53-independent strategy to inhibit MDM2.

Nuclear factor of activated T cells 1 (NFAT1) is the first identified member of the NFAT family. It has well-documented roles in the immune system and inflammatory response (17, 18), but its functions in cancer development and progression are still being elucidated (19, 20). NFAT1 promotes cancer cell proliferation, enhances cell-cycle progression, suppresses apoptosis, induces migration and invasion, and leads to drug resistance through both calcineurin-dependent and -independent pathways (19–22). Therefore, we and others have proposed targeting NFAT1 as a novel anticancer strategy (19, 20). Some compounds have been found to inhibit NFAT1 activity by interfering with the calcineurin activity, but so far, there have been no specific NFAT1 inhibitors developed for cancer therapy (19).

In addition to the individual oncogenic properties of MDM2 and NFAT1, we have recently found that NFAT1 is a novel regulator of the MDM2 oncogene (23). NFAT1 directly binds to the MDM2 P2 promoter, promoting MDM2 transcription (23). Because both MDM2 and NFAT1 have independent oncogenic roles in pancreatic cancer, and based on the identification of the NFAT1-MDM2 pathway, a strategy targeting both MDM2 and NFAT1 may provide a novel treatment for pancreatic cancer. We herein report the identification of MA242 (Fig. 1A), a small molecule that inhibits both MDM2 and NFAT1. The goals of this study were to demonstrate the therapeutic efficacy and safety of MA242 alone and in combination with gemcitabine, and to confirm its mechanisms of action in clinically-relevant models of pancreatic cancer, especially in those deficient in functional p53.

MA242 was synthesized and purified in Dr. Sadanandan E. Velu's laboratory (University of Alabama at Birmingham, Birmingham, AL). The MDM2 siRNA and control siRNA were from Thermo Scientific. For CRISPR/Cas9-mediated MDM2 knockout, the primer sequence of sgRNA-MDM2 was 5′-GTTGGGCCCTTCGTGAGAAT-3′. The primer sequences used for T7EN1 assays and sequencing were as follows: MDM2-clev-forward, 5′-TGCTAGCATTCCTGTGACTGAG-3′; MDM2-clev-reverse, 5′-AAAGCCCTCTTCAGCTTGTGT-3′. A plasmid construct with the GFP-targeted protospacer sequence was used as the negative control. All specific target sequences were amplified and cloned into lenti-CRISPR vectors and verified by DNA sequencing. Antibodies and plasmids were obtained commercially or were provided by other investigators. A detailed list is provided in the Supplementary Materials and Methods.

The human pancreatic cancer HPAC, Panc-1, AsPC-1, Mia-Paca-2, and BxPC-3 cell lines were obtained from the ATCC. The human pancreatic ductal epithelium (HPDE) cell line was a kind gift from Dr. M.S. Tsao (University of Toronto, Ontario, Canada). Cell lines included in the study were validated by analysis of STRs (GenePrint 10 System; Promega) and checked for Mycoplasma contamination by PCR.

All of the in vitro assays used to determine the effects of MA242 alone or combined with gemcitabine on cell viability (24, 25), colony formation (24, 25), cell apoptosis (24, 25), cell-cycle distribution (24, 25), cell migration (25), and cell invasion (26) were performed as described previously. All in vitro assays were performed in triplicate, and all of the experiments were repeated at least three times, with similar results obtained for each replicate (*, P < 0.05; #, P < 0.01).

The protein and mRNA expression levels of MDM2 and other molecules were determined by Western blotting and real-time quantitative PCR, respectively (27). The primer sequences used for the amplification of genes were as follows: MDM2 sense, 5′-ATCATCGGACTCAGGTACA-3′; MDM2 antisense, 5′-GTCAGCTAAGGAAATTTCAGG-3′; CCNE1 sense, 5′-TCT GGA TTG GTT AAT GGA GGT-3′; CCNE1 antisense, 5′-TGG TGC AAC TTT GGA GGA-3′; GAPDH sense, 5′-GGAGTCCACTGGCGTCTTCAC-3′; GAPDH antisense, 5′-GAGGCATTGCTGATGATCTTGAGG-3′. Immunofluorescence staining was performed to determine the expression and location of the MDM2 protein in the cells (24). The cytoplasmic and nuclear fractions were extracted from MDA242-treated cells using the NE-PER nuclear and Cytoplasmic Extraction Kit (Pierce). The MDM2 promoter activity was determined using a luciferase reporter assay system (Promega; ref. 23). Briefly, cells were transfected with MDM2 P1 or P2 (full-length, deleted, or mutated) luciferase plasmids with Renilla luciferase reporter as an internal control for 12 hours, followed by treatment with various concentrations of MA242 for 24 hours. The degradation rates of proteins and mRNAs were examined using pulse-chase assays (27).

The cells were treated with MA242 in the presence or absence of ionomycin (4 μmol/L), an activator of calcineurin, for 6 hours, then the nuclear fractions were extracted using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific; ref. 23). The prepared nuclear extracts were preincubated with 1 μg of poly-(dI:dC) (Thermo Scientific) and then reacted with a biotin-labeled MDM2 probe at room temperature for 30 minutes. The sequences of the biotin-labeled probes were: MDM2 forward, 5′-GCAGGTTGACTCAGCTTTTCCTCTTGAGCTGGTCAAGTTCA-3′ and MDM2 reverse, 5′-TGAACTTGACCAGCTCAAGAGGAAAAGCTGAGTCAACCTGC-3′.

The cells were treated with DMSO as a vehicle control or with MA242 for 6 hours, then the chromatin immunoprecipitation (ChIP) assay was performed as reported previously (23). The specific primer pairs used were: 5′-CCCCCGTGACCTTTACCCTG-3′ and 5′-AGCCTTTGTGCGGTTCGTG-3′ for qualitative or quantitative PCR amplification of the responsive element of the MDM2 promoter.

The animal protocols were approved by the Institutional Animal Use and Care Committee. Female 4- to 6-week-old athymic nude mice (nu/nu, 4–6 weeks) were obtained from Charles River Laboratories International, Inc. Two pancreatic cancer cell lines, AsPC-1-Luc and Panc-1-Luc, were used in this study (24). Briefly, 30 μL of AsPC-1-Luc or Panc-1-Luc cell solution (1 × 10⁶ cells in PBS) was injected into the head of the pancreas. MA242 was dissolved in PEG400:ethanol:saline (57.1:14.3:28.6, v/v/v). The treatment was carried out in the following way: The control group received the vehicle only. For AsPC-1 tumor-bearing mice, MA242 was administered by intraperitoneal injection at a dose of 10 mg/kg/day, 5 day/week for 3 weeks. For Panc-1 tumor-bearing mice, MA242 was administered by intraperitoneal injection at a dose of 2.5 or 5 mg/kg/day, 5 day/week for 5 weeks. Gemcitabine (Selleckchem) was administered at a dose of 20 mg/kg on days 1, 3, and 5 every week for 2 weeks. The combination group received MA242 at a dose of 5 mg/kg, 5 day/week for 5 weeks and gemcitabine at a dose of 20 mg/kg on days 1, 3, and 5 every week for 2 weeks. At the end of the experiments, all mice were examined for tumor metastasis to various organs, tumors and various tissues were removed for Western blotting and pathology evaluation (24, 25).

Briefly, tumors and various tissues were removed from mice, fixed in 10% formalin, and embedded in paraffin. The tumor and tissue sections (5 μm thick) were then prepared, deparaffinized, rehydrated, and washed (24, 25). The IHC staining of target proteins was performed using biotinylated antibodies and then the sections were counterstained with hematoxylin, mounted, and analyzed. The in vivo apoptosis was measured using a TUNEL Staining Kit (Trevigen) according to the manufacturer's instructions (24, 25).

The data were analyzed using the Prism software program version 6 (GraphPad Software Inc.). Two-sided t tests were used for comparisons between two groups. All data are expressed as the means ± SEM from at least three independent experiments. P < 0.05 was considered to be statistically significant.

Thus far, there have been no specific MDM2 and NFAT1 dual inhibitors available. We were motivated to identify bifunctional small molecules that could bind to the both MDM2 and NFAT1, promoting MDM2 degradation and inhibiting NFAT1-mediated MDM2 transcription, respectively. By using high-throughput virtual and cell-based screening assays, we discovered a class of makaluvamine analogs with the desired properties. To improve the activity and specificity of makaluvamines, we further designed and synthesized a class of makaluvamine analogs (data not shown). One of these analogs, MA242 (Fig. 1A), was identified as a potent and selective MDM2 and NFAT1 dual inhibitor in a molecular modeling study, Biacore assay, and pull-down assays (data not shown). As shown in Supplementary Fig. S1A and S1B, at a concentration of 0.1 μmol/L, MA242 induced a significant reduction of the MDM2 and NFAT1 expression in Panc-1 cells, indicating that it was the most potent of the new compounds. Similar results were obtained in other pancreatic cancer cell lines (data not shown).

To assess its activity and selectivity, MA242 was examined for effects on cell viability in a normal pancreatic HPDE cell line [p53 wild-type (wt)] and a panel of MDM2-overexpressing pancreatic cancer cell lines, including HPAC (p53 wt), Panc-1 [p53 mutant (mt)], AsPC-1 (p53 null), Mia-Paca-2 (p53 mt), and BxPC-3 (p53 mt) (28). As shown in Fig. 1B, MA242 significantly inhibited pancreatic cancer cell growth, with IC₅₀ values ranging from 0.1 to 0.4 μmol/L, regardless of the p53 status of the cells. However, MA242 showed minimal effects on the growth of normal HPDE cells (IC₅₀ = 5.81 μmol/L), indicating that MA242 had selective effects against cancer cells. MA242 was further evaluated for in vitro anticancer activity and the specificity of its inhibition of MDM2 and NFAT1 in pancreatic cancer cells with different p53 backgrounds, including the HPAC, Panc-1, and AsPC-1 cell lines. MA242 effectively inhibited the cell colony formation (Fig. 1C), slightly arrested cells at G₂–M phase (Fig. 1D), and induced apoptosis (Fig. 1E) in all three cell lines in a concentration-dependent manner, regardless of the p53 status of the cells, indicating that apoptosis was the major regulatory mechanism for MA242-induced cell death. In addition, MA242 significantly decreased the MDM2 and NFAT1 protein levels at a low concentration in all three cell lines (Fig. 1F). However, MA242 did not affect the protein expression levels of cyclin E in all three cell lines, indicating that the compound did not nonspecifically affect other inducible proteins. We also observed that when MDM2 was inhibited by MA242, there was activation of p53 signaling in HPAC (p53 wt) cells, but no effect on the expression levels of p53 in the Panc-1 (p53 mut) and AsPC-1 (p53 null) cells. MA242 also increased the expression levels of p21 and cleaved PARP in a p53-independent manner.

To confirm that MA242 exerts p53-independent antipancreatic cancer activity in vivo, we next examined its efficacy in Panc-1 and AsPC-1 orthotopic tumor models. As shown in Fig. 2A, the nude mice bearing Panc-1 orthotopic tumors were treated with vehicle or MA242 (2.5 and 5 mg/kg/day, 5 days/week) by intraperitoneal injection for 5 weeks, resulting in 56.1% and 82.5% inhibition of tumor growth, respectively (Fig. 2A1–2). In the AsPC-1 orthotopic model, mice were treated with MA242 at 10 mg/kg/day, 5 days/week for 3 weeks. MA242 significantly suppressed the growth of AsPC-1 orthotopic tumors by 89.5% (P < 0.01) compared with the tumors in control animals (Fig. 2B1–2). MA242 treatment led to almost complete tumor regression in MD242-treated mice in both models. Importantly, there were no significant differences in the average body weights between the vehicle- and MA242-treated mice in either of the models, suggesting that MA242 did not have significant host toxicity at these effective doses (Figs. 2A3 and B3). At the end of the experiments, the major organs (liver, lungs, kidneys, spleen, heart, and brain) were dissected from mice bearing AsPC-1 orthotopic tumors for histologic examinations, and no tumor metastasis or organ abnormalities were observed in control or treatment groups (Supplementary Fig. S2), confirming the efficacy and safety of the treatment.

To examine the effects of MA242 on MDM2 and NFAT1 in vivo, we evaluated the protein expression levels of MDM2 and NFAT1 in both Panc-1 and AsPC-1 orthotopic tumors. As shown in Fig. 2C, the IHC staining of tumor tissues indicated that the MDM2 and NFAT1 expression levels were significantly reduced in MA242-treated tumors. TUNEL staining of the tumor tissues demonstrated that there was a significant increase in apoptosis in the MA242-treated tumors, compared with that observed in vehicle-treated tumors. These results were confirmed by a Western blotting analysis (Fig. 2D). Consistent with the in vitro results, MA242 decreased the expression of MDM2 and NFAT1, and increased the expression levels of p21 and cleaved-PARP in both AsPC-1 and Panc-1 orthotopic tumors, compared with the tumors from control animals.

Overcoming gemcitabine resistance in pancreatic cancer cells remains a major challenge to the effective treatment of the disease (29). MA242 was further examined for its effects on the sensitivity of gemcitabine-resistant HPAC, Panc-1, and AsPC-1 cell lines (30) to gemcitabine. As shown in Fig. 3A, treatment with MA242 at a low concentration (0.05 μmol/L) significantly increased the gemcitabine sensitivity in all three cell lines, with remarkably reduced IC₅₀ values for gemcitabine in all of these cell lines. Furthermore, gemcitabine induced more apoptosis in pancreatic cancer cells that were treated with MA242 than was observed in the vehicle-treated cells (Fig. 3B).

To determine whether MA242 sensitizes pancreatic cancer cells to chemotherapy in vivo, the effects of gemcitabine (20 mg/kg, intraperitoneal injection on days 1, 3, and 5/week for 2 weeks) were evaluated alone or in combination with MA242 (5 mg/kg) in the Panc-1 orthotopic tumor-bearing mice. As shown in Fig. 3C1 and 2, gemcitabine alone exerted only moderate inhibitory growth effects, with tumor growth inhibited by about 47.8%, whereas the combination of gemcitabine with MA242 led to a complete tumor regression in almost all of the mice (94.1% inhibition). Of note, there were no significant changes in the average body weight in the MA242 and gemcitabine combination-treated mice compared with control mice (Fig. 3C3), indicating that MA242 and gemcitabine combination was safe at the effective therapeutic doses.

The propensity of pancreatic cancer for early metastasis, with frequent involvement of the peritoneum, liver, and lungs, is one the major reasons for the low 5-year survival rate (∼6%) in patients (6). Therefore, we next examined the effects of MA242 on cell migration and invasion with Panc-1 and AsPC-1 cell lines, which are invasive and metastatic (24). As shown in Fig. 4A, the Panc-1 and AsPC-1 cells both migrated into the entire wounded area by 48 hours, whereas treatment with MA242 at low concentrations (0.05 and 0.1 μmol/L) significantly inhibited the cell migration. Similarly, at a concentration of 0.1 μmol/L, MA242 reduced the invasion of Panc-1 and AsPC-1 cells by 75.8% and 77.0%, respectively, compared with the control (Fig. 4B).

MA242 was further examined for effects on metastasis in mice bearing Panc-1 orthotopic tumors. Necropsy showed that 10 of 11 mice treated with the vehicle developed metastatic lesions in the peritoneum, whereas the incidence of peritoneal dissemination in the mice treated with 2.5 and 5 mg/kg/d MA242 was 4/11 and 3/11, respectively (Fig. 4C and D). Compared with the vehicle-treated mice, MA242 treatment also significantly inhibited the metastasis to the liver and lungs in a dose-dependent manner (Figs. 4C–E). Of note, 5, 5, and 1 of 12 mice treated with gemcitabine were found to have metastatic lesions in the peritoneum, liver, and lungs, respectively, whereas only 1 of 11 mice treated with the combination of gemcitabine and MA242 had liver metastasis, and none of these mice had peritoneal or lung metastases (Figs. 4C–E). These results were confirmed by histopathologic examinations of the liver and lung tissues from mice in the control and treatment groups (Supplementary Fig. S3A). The metastatic cells in livers and lungs were further confirmed using IHC staining of specific pancreatic cancer biomarkers CA19-9 and cytokeratin 7 (Supplementary Fig. S3B). No metastasis or abnormalities were found in any other major organs (kidneys, spleen, heart, and brain) from the treated mice, indicating the absence of any overt host toxicity (Supplementary Fig. S3A).

We next examined MA242′s effects on the turnover of the MDM2 protein in the HPAC and Panc-1 cell lines. As shown in Fig. 5A, MA242 shortened the half-life of MDM2 in both cell lines. This protected wild-type p53 from MDM2-mediated degradation in the HPAC cells, but there was no significant effect of MA242 on mutant p53 in the Panc-1 cells. Further, the MA242-induced MDM2 inhibition was reduced by treatment of the cells with a proteasome inhibitor, MG-132 (Fig. 5B), indicating that MA242 promoted the proteasomal degradation of MDM2. These results were confirmed by MDM2 ubiquitination assays in both HPAC and Panc-1 cells (Fig. 5C).

To determine if MA242 specifically induces MDM2 self-ubiquitination, its effects on the expression of wild-type MDM2 and an MDM2 mutant (C464A) lacking ubiquitin E3 ligase activity were examined. As shown in Fig. 5D, MA242 induced the degradation of wild-type MDM2, but it had no significant effect on this mutant MDM2. An immunofluorescence study was then performed, which showed that the degradation of both nuclear and cytoplasmic MDM2 was increased by MA242 treatment (Fig. 5E). The similar results were confirmed by Western blot analysis (Fig. 5F).

As shown in Fig. 6A, MA242 concentration-dependently inhibited MDM2 mRNA expression; however, it did not affect the stability of the MDM2 mRNA (Supplementary Fig. S4A). The compound also did not affect the mRNA expression of CCNE1, indicating a specificity of MA242′s inhibitory effects on MDM2 mRNA expression (data not shown). Further, MA242 inhibited the MDM2 P2 promoter activity in a concentration-dependent manner, but not MDM2 P1 promoter activity (Fig. 6B).

Several transcription factors have been reported to regulate MDM2 transcription via the P2 promoter (31). To determine which transcription factor was involved in the MA242-induced inhibition of MDM2 transcription, various deletions (Supplementary Fig. S4B) and site mutations of the MDM2 P2 promoter were transfected into HAPC and Panc-1 cells, followed by treatment with MA242. The results showed that the shortest deletion, Luc 03, retained the responsiveness to MA242 (Fig. 6C; Supplementary Fig. S4C). Next, we made site mutations of several transcription factor binding sites in this segment, including those for MEF2, NFAT1, AP1, and ETS. The results showed that mutation of the NFAT1 site significantly reduced the effects of MA242 on the P2 promoter activity, indicating that MA242 could inhibit NFAT1-mediated MDM2 transcription (Fig. 6D; Supplementary Fig. S4D). The results were confirmed using MDM2 P2 promoters with double mutations (ΔAP1–ETSα) and triple mutations (ΔAP1–ETSα–NFAT; Fig. 6E; Supplementary Fig. S4E).

Further studies were performed to determine how MA242 inhibits NFAT1-mediated MDM2 transcription. Because NFAT1 activates MDM2 transcription via direct binding to the MDM2 P2 promoter (23), EMSA and ChIP assays were utilized to examine the effects of MA242 on the NFAT1–MDM2 P2 promoter complex. As shown in Fig. 6F, MA242 inhibited the ionomycin (an activator of the calcineurin–NFAT pathway)-enhanced NFAT1–MDM2 P2 promoter binding in HPAC and Panc-1 cells. Similar results were further confirmed by the ChIP assay (Fig. 6G).

To demonstrate the importance of MDM2 for the sensitivity of the pancreatic cancer cells to MA242 treatment, MDM2 knockdown (KD) experiments using the CRISPR/Cas9 technique were performed in Panc-1 cells. As shown in Fig. 7A, the MDM2 KD stable cell line appeared approximately 92% knockdown of MDM2 protein expression. Treatment of the parental cells with MA242 inhibited the expression of NFAT1 and induced the expression of cleaved-PARP in a concentration-dependent manner. Compared with these control Panc-1 cells, MDM2 KD itself caused 20% inhibition of cell growth (Fig. 7B) and two-fold increase in apoptosis (Fig. 7C). In addition, the MDM2 KD cells showed a decreased response to MA242, leading to decreased effects of MA242 on the growth and apoptosis of the cells (Fig. 7B and C). Similar results were obtained in HPAC and Panc-1 cells with siRNA-mediated MDM2 KD (Supplementary Fig. S5A–S5F). To demonstrate that MA242-induced MDM2 degradation causes apoptosis but MA242-induced apoptosis does not cause MDM2 degradation, we further determined the effects of MA242 on MDM2 expression and cell apoptosis in the presence or absence of an apoptosis inhibitor z-VAD-FMK. As shown in Supplementary Fig. S6A and S6B, z-VAD-FMK significantly reduced MA242-induced cell apoptosis but it did not affect MA242′s inhibitory effects on MDM2, indicating that MA242 caused MDM2 degradation, leading to cell apoptosis.

The aim of this study was to develop a safe and effective dual MDM2 and NFAT1 inhibitor for pancreatic cancer therapy. MDM2 has been demonstrated to be a “druggable” target because it was discovered to be the major negative regulator of the p53 tumor suppressor (7–12), and a variety of strategies have been developed to inhibit its expression and/or functions. Several MDM2 inhibitors are currently in clinical trials (14), but these inhibition strategies have proven to be challenging for several reasons. First, wild-type p53 is necessary for all inhibitors that target the MDM2–p53 interaction, but p53 mutation is a common genetic event in human cancers. Second, other than the p53 binding site, it has proven difficult to identify other active binding sites for small molecules on MDM2. Third, specific inhibition of MDM2 expression and/or functions by these inhibitors leads to a hyperactive compensation of MDM2 through other pathways, such as p53-induced feedback-mediated increases in MDM2. Therefore, the therapeutic efficacy of the current MDM2 inhibitors will likely be limited in human cancers, especially in those with p53 deficiency. New and more effective strategies for targeting MDM2 are needed.

It has also been shown that NFAT1 is overexpressed in malignant pancreatic ductal adenocarcinomas compared with normal adjacent tissues, and is associated with advanced stages of the disease (21). Therefore, we and others have proposed NFAT as a target for cancer therapy. Indeed, a few calcium channel blockers (e.g., CsA and tacrolimus) have been found to inhibit NFAT1 activity and showed promising anticancer activity in the preclinical setting that was postulated to be related to effects on calcineurin signaling. However, due to the lack of specificity of these drugs, patients on long-term immunosuppressive treatments actually exhibit increased rates of cancer incidence, neuro- and nephrotoxicity, as well as cardiovascular and diabetic complications (20). Therefore, new treatment strategies that specifically inhibit NFAT1 activity are needed.

Most of the previously developed anticancer SMIs specifically target one gene in one pathway. However, due to the presence of pathway convergence and crosstalk, the exclusive inhibition of one target often leads to the hyperactivation of other pathways, as well as limited efficacy in clinical trials. In this study, we have identified MA242 as a new dual inhibitor of MDM2 and NFAT1 based on virtual structural screening and physical–chemistry evaluations. MA242 is distinct from previous single-pathway inhibitors; it exhibits dual inhibition of both MDM2 and NFAT1, leading to a profound reduction of both the MDM2 and NFAT1 levels in pancreatic cancer cells, regardless of the p53 status. Our results demonstrated that MA242 increased MDM2 self-ubiquitination and promoted its proteasomal degradation. In addition, MA242 enhanced NFAT1 protein degradation, resulting in the repression of MDM2 transcription. Most importantly, the MA242-induced anticancer activity is dependent on MDM2 inhibition. In the present study, in order to validate that MDM2 is the major target of MA242, we used both CRISPR/Cas9 gene editing and siRNA techniques. We confirmed that knockdown of MDM2 was toxic to the cells and demonstrated that knockdown of MDM2 resulted in decreased effectiveness of the compound MA242, supporting the importance of MDM2 inhibition in the MA242-induced antipancreatic cancer activities.

Considering that resistance to conventional therapies and early metastasis are the major causes of pancreatic cancer mortality, and p53 mutations were largely responsible for the limited efficacy of MDM2 inhibitors in previous studies (29, 32), we used a variety of pancreatic cancer models to evaluate the translational potential of MA242. First, normal pancreatic cells and a panel of pancreatic cancer cell lines with various backgrounds of p53 (wt, mt, and null) were used to demonstrate the cancer cell selectivity and MDM2 inhibitory effects of MA242. HPAC cells showed different responses to MA242 treatment in MTT assay and colony formation assay, which indicated that MA242 might mainly inhibit the cell proliferation but not cell metabolic activity. In addition, the p53-induced feedback increase in MDM2 could be partially responsible for this discrepancy. Second, the in vivo efficacy and safety of MA242, and its p53-independent effects, were examined in both AsPC-1 and Panc-1 orthotopic mouse models. We first demonstrated the in vivo efficacy of MA242 in a Panc1 orthotopic model at 2.5 and 5 mg/kg because we did not observe any host toxicity at these doses in our initial safety studies (data not shown). To further demonstrate the in vivo efficacy and safety of MA242, we assessed the effects of the compound at a higher dose (10 mg/kg) in an AsPC1 orthotopic model. These results indicated that MA242 at 10 mg/kg is a safe and effective dose for treating human pancreatic cancer in vivo. Third, the effects of MA242 on cell migration and invasion were examined in pancreatic cancer cell lines with invasive and metastatic properties; the in vivo efficacy against tumor metastasis was also evaluated. Fourth, three gemcitabine-resistant cell lines were utilized to demonstrate the MA242-induced chemosensitization of pancreatic cancer cells to gemcitabine in vitro, whereas one of these cell lines was further used to develop an orthotopic mouse model for in vivo evaluation. Although gemcitabine is a first-line treatment for patients with locally advanced or metastatic pancreatic cancer, a large number of patients with pancreatic cancer do not respond to gemcitabine due to the high levels of intrinsic and acquired chemoresistance. Gemcitabine can interrupt DNA synthesis and then cause cell death by apoptosis (33). Several signaling pathways playing critical roles in the development of gemcitabine chemoresistance have been reported. Genetic or epigenetic alterations regulating gemcitabine-induced apoptosis, such as mitogen activated protein kinase p38 (p38MAPK), p53, histone deacetylase (HDAC), and ceramide, appear to impact pancreatic cancer chemosensitivity directly or indirectly (34–37). In this study, we observed a p53-independent apoptosis induction on the treatment of the cells with the gemcitabine treatment, which was significantly altered by the combination of MA242. As examples of the potential p53-dependent effects of combination treatment, p53 activation by MA242 and gemcitabine stimulates a wide network of signals that act through both extrinsic and intrinsic apoptotic pathways, including the activation of a Caspase cascade and the shifting the balance in the Bcl-2 family towards the pro-apoptotic members. In p53 nonfunctional cells, it is possible that MA242 and gemcitabine treatment causes other well-known apoptosis mediators directly. Gemcitabine induces the activation of acidic sphingomyelinase (SMase) and increases the production of ceramide, resulting in apoptosis induction (37). MA242 may actively or physically bind to other MDM2-related apoptosis mediators such as p73 and FOXO3a (38), leading to subsequent induction of apoptosis. In the present report, our combination therapy significantly improved the efficacy of gemcitabine alone, demonstrating chemosensitization of gemcitabine-resistant cells in vitro and in vivo. However, the mechanisms underlying MA242-mediated chemosensitization are still not fully understood. Further studies are needed to explore the molecular mechanisms by which MA242 increases chemosensitization.

In summary, MA242 is a potent and selective dual inhibitor of MDM2 and NFAT1 with distinct mechanisms of action, compared with the existing MDM2 or NFAT1 inhibitors. We have also shown that MA242 has promising efficacy both alone and in combination with gemcitabine in several preclinical models of pancreatic cancer. Our data suggest that the pharmacologic inhibition of both MDM2 and NFAT1 is a promising strategy for the treatment of this disease, even in tumors lacking functional p53. MA242 warrants further evaluation as a new therapeutic option for pancreatic cancer to be used alone and/or in combination with gemcitabine.

No potential conflicts of interest were disclosed.

Conception and design: W. Wang, J.-J. Qin, S. Voruganti, R. Zhang

Development of methodology: W. Wang, J.-J. Qin, S. Voruganti, B. Nijampatnam, S.E. Velu, R. Zhang

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): W. Wang, J.-J. Qin, S. Voruganti, S.E. Velu, R. Zhang

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): W. Wang, J.-J. Qin, S. Voruganti, B. Nijampatnam, S.E. Velu, R. Zhang

Writing, review, and/or revision of the manuscript: W. Wang, J.-J. Qin, S. Voruganti, B. Nijampatnam, K.-H. Ruan, M. Hu, J. Zhou, R. Zhang

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): W. Wang, J.-J. Qin, S. Voruganti, B. Nijampatnam, S.E. Velu, M. Hu, R. Zhang

Study supervision: W. Wang, R. Zhang

This work was supported by the NIH/NCI grants (R01 CA186662 and R01CA214019). The content is solely the responsibility of the authors, and does not necessarily represent the official views of the NIH. W. Wang was also supported by American Cancer Society (ACS) grant RSG-15-009-01-CDD. S.E. Velu was supported by Collaborative Programmatic Development grant 1UL1RR025777 from the UAB Comprehensive Cancer Center and NIH National Center for Research Resources. R. Zhang was also supported by funds for Robert L. Boblitt Endowed Professor in Drug Discovery and research funds from College of Pharmacy and University of Houston. We thank Dr. Elizabeth Rayburn for excellent assistance in the preparation of this manuscript.

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